DE60214127T2 - HYBRIDOMY CELL LINE G250 AND ITS USE FOR THE PRODUCTION OF MONOCLONAL ANTIBODIES - Google Patents

Abstract

The invention relates to novel nucleic acid sequences which encode an antibody suitable in the field of tumor diagnostics and therapeutics. Further, a method of producing recombinant antibodies is provided, wherein the novel nucleic acid sequencs are employed.

Description

The The invention relates to the hybridoma G250 or a progeny cell of it, the G250 antibodies can produce.

The first by Kohler and Milstein in Nature, 256, 495-997 (1975) immunized fusion of mouse myeloma cells with spleen cells mice allows it to get continuous cell lines, the homogeneous antibodies, so called monoclonal antibodies (mAb), produce.

It Several examples are known in which hybrid cells or hybridomas to be discribed. These hybridomas are used to make antibodies, the for Various scientific studies are useful (Current Topics in Microbiology and Immunology, Vol. 81 - "Lymphocyte Hybridomas", F. Melchers et al., Springer-Verlag (1978) and references therein; C.J. Barnstable et al., Cell, (1978), 14, 9-20; P. Parham, W.F. Bodmer, Nature (1978), 276, 397-399; Handbook of Experimental Immunology, 3rd Edition, Volume 2, D.M. Wier, Editor, Blackwell, 1978, Chapter 25, Chem. Eng. News, 15-17 (1979); Kennett, R.H., McKearn, J.T. and Bechtol, K.B. (1980) Monoclonal Antibodies. Hybridomas: A New Dimension in Biological Analysis (Plenum, New York)). These publications describe the basic techniques for the production of monoclonal antibodies using Hybridomas.

Of the monoclonal antibodies G250, subclass IgG1, recognizes an antigen that is preferential on Membranes of cells in renal cell carcinoma (RCC) is expressed and not expressed in the normal epithelium of the proximal tubule becomes. The antibody G250 was engineered by immunizing a mouse with cell homogenates primary RCC lesions obtained from different patients (Oosterwijk et al., Int. J. Cancer 38 (1986), 489-494).

Of the monoclonal antibodies G250 as well as chimera Derivatives thereof have been used in clinical studies (Steffens et al., J. Clin. Oncol. 15 (1997), 1529-1537). The nucleic acid sequence, the for the antigen-binding site encoded by G250 is the subject of the same time pending U.S. Application No. 60/266,853, incorporated herein by reference.

The Preparation of a hybridoma cell line expressing the G250 antibody, has been generally disclosed in International Patent Application WO 88/008854 and in Oosterwijk et al. (supra). As described above, was used as an immunogen a cell homogenate from primary RCC lesions consisting of various Patients were received, and thus used a non-specific Material. in addition, the hybridoma cell line did not become one after the Budapest Deposited by a recognized depositary. As a result, it seems not possible, the hybridoma cell line G250 using the publicly available Exactly rework documents of the prior art.

meanwhile It was found that the antibody G250 to the so-called MN antigen binds, the e.g. in WO 93/18152. The G250 binding site to the However, MN antigen is still unknown. In addition, recent results that the G250 binding site is a conformational epitope, so that it is even more difficult to rework the G250 hybridoma cell line as no specific epitope sequence on the MN antigen, which is G250 binds, can be provided.

The The present invention thus relates to a hybridoma cell containing the monoclonal antibody G250 can produce. This hybridoma cell was named after the Budapest Contract for the deposit of microorganisms on 11 September 2001 at the German Collection of Microorganisms and Cell Cultures GmbH (DSMZ), Mascheroder Weg 1b, 38124 Braunschweig, Germany, under the accession number DSM ACC 2526 deposited. The deposit represents the first publicly available Revelation of a G250 antibody producing hybridoma cell line.

One Another aspect of the invention is one of the hybridoma DSM ACC 2526 derived progeny cell producing a G250 antibody.

In a preferred embodiment, the present invention relates to a progeny cell of the deposited G250 antibody-producing hybridoma cell, wherein the progeny cell is recombinantly DNA-sequestered, for example by transferring genetic material encoding the G250 antibody or at least an antigen-binding site thereof. into a receptor cell. The genetic material can be obtained directly or indirectly from the deposited hybridoma G250. "Received directly" means that the G250 genetic material is derived from the deposited hybridoma cell. "Indirect "means that the G250 genetic material is derived from an already existing G250 progeny cell or from another source of output, including chemical synthesis.

Preferably, the G250 genetic material comprises nucleotide sequences encoding at least the G250 antigen binding site, in particular nucleotide sequences encoding the complementarity determining regions CDR3, CDR2 and / or CDR1 of the antigen binding site on the heavy chain, as in 1 (as H1-H3) and / or encode the complementarity determining regions CDR3, CDR2 and / or CDR1 of the antigen binding site on the light chain, as in 1 shown (referred to as L1-L3).

According to the invention, the term "G250 antibody" includes any antibody including multispecific antibodies (eg bispecific antibodies) and antibody fragments as long as they have the desired activity, ie as long as they have at least one G250 antigen binding site. IgG (eg: IgG ₁ , IgG ₂ , IgG ₃ or IgG ₄ ), IgD, IgA or IgE antibodies, in particular an IgG antibody, a recombinant antibody or an antibody fragment, which is produced by proteolytic methods or by recombinant DNA Procedure has been obtained.

Of the Term "monoclonal Antibody "is used herein to an antibody to designate that of a population of substantially homogeneous antibodies is obtained. This means that the individual antibodies within the population are identical except for possible, naturally occurring mutations present in small quantities could be.

Of the Term "antibody" is used herein to designate a polypeptide having at least one G250 antigen binding site includes, i. at least the CDR3 region of the G250 heavy chain and / or the CDR3 region of the G250 light chain or a variant of the G250 CDR3 region, which has one compared to the original G250 CDR3 region identity at the amino acid level of at least 80%, preferably of at least 90%, with the proviso that the variant of the CDR3 region has the equivalent antigen binding properties and in particular the same affinity and specificity in comparison to the original CDR3 region having.

The term "antibody" herein preferably includes chimeric antibodies, humanized and fully humanized antibodies, single-chain antibodies, eg sFv antibody fragments, bispecific fragments, proteolytic or recombinant antibody fragments, such as Fv, Fab, Fab 'or F ( ab ') ₂ fragments or other antigen-binding partial sequences of the antibodies The antibody can also be a fusion antibody or form a conjugate with other units.

The Antibodies described herein include in particular chimera Antibody, in which part of the heavy and / or light chain that the Comprises antigen binding site that is identical or homologous to the corresponding sequences, from the original ones Hybridoma cell line G250, while the remainder of the Chains is identical or homologous to corresponding sequences, that are descended from other species or that belong to a different class of antibodies or subclass, or fragments of such antibodies, as long as they persist the desired biological activity exhibit. Preferably, the chimeric antibody comprises variable regions, e.g. the complementarity determinant Regions (CDRs) and the scaffolding regions heavy chain and light chain of the original one G250 monoclonal antibody and the constant human sequences, preferably the constant ones human sequences of kappa light chain and gamma heavy chain. The production of chimeras antibody is e.g. in Morrison et al. (Proc Natl Acad Sci USA 81 (1984), 6851-6855) described herein incorporated by reference.

Furthermore, the antibodies described herein include, in particular, humanized antibodies or fully humanized antibodies. Humanized antibodies are immunoglobulins, immunoglobulin chains or fragments thereof containing a short sequence derived from non-human immunoglobulin. In particular, humanized antibodies are human immunoglobulins in which residues of a CDR of a particular human antibody are replaced with residues of the G250 CDR, particularly the CDR1, 2 and / or 3 region of the heavy and / or light chain. In addition, humanized antibodies may include residues that are not found in either the human recipient antibody or the introduced G250 CDR sequences. These changes are being introduced to refine and optimize antibody properties. In general, the humanized or fully humanized antibody comprises substantially all of the at least 1 and usually 2 variable domains, all or substantially all of the CDR regions corresponding to the regions of the original G250 immunoglobulin, and all or substantially all of the domains all framework regions and constant regions corresponding to human immunoglobulin sequences. The production of humanized antibodies is described, for example, in Jones et al. (Nature 321 (1986), 522-525), Riechmann et al. (Nature 332 (1988), 323-329 and Presta (Curr Op., Struct. Biol., 2: 322 (1992), 332-339), which are incorporated herein by reference.

Farther include the antibodies in particular single-chain antibodies, such as single-chain Fv antibody fragments, comprising the VH and VL domains an antibody, these domains in a single polypeptide chain. Generally included the Fv polypeptides also have a polypeptide linker between the VH and the VL domains, so that the sfv the desired Structure for antigen binding can take. The production of sFv antibodies e.g. in Plückthun in: The Pharmacology of Monoclonal Antibodies, Volume 113, Rosenburg and Moore, Eds., Springer Verlag, NY, pp. 269-315 (1994), Barbas III (Methods: Companion Methods Enzymol. 2 (1991), 119) and Hoogenboom et al. (Immunol., Rev., 130 (1992), 41-68), incorporated herein by reference Reference is made.

Farther include the antibodies in particular bispecific antibodies, i.e. small antibody fragments, which have two antigen binding sites, these fragments a variable domain heavy chains contained in the same polypeptide chain with a variable domain the light chain is connected. By using a linker, too short to match the two domains on the same chain enable, have to the domains with the complementary ones domains pairing another chain so as to have two antigen binding sites form. The production of bispecific antibodies is e.g. in Hollinger et al. (Proc Natl Acad Sci USA 90 (1993), 6444-6448), to which reference is made herein.

Farther include the antibodies in particular multispecific antibodies, e.g. heterobispecific antibodies that at least one G250 antigen binding site and an antigen binding site another antibody, e.g. an anti-CD3 antibody.

Of the Antibody, that of the G250 hybridoma cell or a progeny cell thereof can be produced with a Macker or an effector component be merged or linked. For example, the G250 antibody may be on be recombinantly modified so as to bind to cytokines, such as interleukin-2 (IL-2), to tumor necrosis factor (TNF) and / or granulocyte macrophage colonies Factors (GM-CSF) is bound.

In addition, the G250 antibody may be conjugated, eg, by covalent coupling or fusion to a suitable labeling group, eg, a fluorescent group, a radioactive label group, etc., or to a cytotoxic agent comprising radioactive isotopes, such as I, Y, Pr , Tm, In, such as ¹²³ I, ¹²⁵ I, ¹³⁵ I, ¹¹¹ In or ¹¹¹ In, chemotherapeutic agents and toxins, such as enzymatically active toxins derived from bacteria, fungi, plants or animals, or fragments thereof.

The Receptor cell containing the G250-specific genetic material the original one G250 hybridoma cell or a progeny cell thereof or synthetic Origin transmitted may be any host cell expressing antibodies can. For example, the host cell may be a prokaryotic, preferably an E. coli cell, or a eukaryotic cell. preferred eukaryotic cells are e.g. Insect, yeast and mammalian cells. Particularly preferred host cells are mammalian cells, e.g. humane Myeloma cells or mouse myeloma cells or CHO cells.

It also includes the present invention a method for producing a G250 antibody or Derivatives thereof, comprising: culturing a G250 hybridoma cell or a progeny cell thereof under suitable conditions which is a G250 antibody and recovering the antibody and / or derivative thereof from the cell and / or from the cell culture medium.

Of the according to this antibody obtained in the present method is particularly suitable for the preparation of pharmaceutical formulations containing these antibody as an active ingredient, together with pharmaceutically acceptable carriers, diluents and / or aids.

In a particularly preferred embodiment, the active ingredient is a chimeric G250 antibody, either as such or as a conjugate with a radioactive group, eg ¹²³ I, ¹²⁵ I, ¹³¹ I, ^99m Tm or ¹¹¹ In.

Moreover, the present invention relates to the use of the deposited hybridoma cell and Progeny cells thereof for the production of G250 antibodies as described above, eg of monoclonal antibodies, chimeric antibodies, humanized antibodies, fully humanized antibodies, bispecific antibodies and antibody fragments, such as (Fab ') ₂ , Fab', Fab and Fv fragments.

Of the chimera G250 antibody was in clinical trials in treatment after surgery used successfully by patients with renal cell carcinoma. Even though the studies are not completed at the present time, show the treated patients already have a significant increase in average chances of survival (over 15 Months) compared to untreated patients (3 months) or to patients treated with standard care (10-12 Months).

Surprisingly was found that in some cases more than six months after the beginning of the therapy a tumor decrease had occurred. Therefore is the chimera G250 antibody and other G250 antibodies able to be a delayed Immune response in cancer treatment, especially in the treatment of renal cell carcinoma and particularly preferred in the treatment Metastases after tumor surgery.

example 1

Deposit of the G250 hybridoma cell line

The G250 hybridoma cell line was prepared according to example 1 from WO 88/08854. This will become a general immunization protocol described. Further information, e.g. Information about the molecular properties of the G250 antibody and the G250 hybridoma cell, are not included.

The G250 hybridoma cell line was established on September 11, 2001 as required of the Budapest Treaty with the DSMZ with the approval number (accession number) DSM ACC 2526 deposited.

Example 2

Mapping of monoclonal antibody G250 recognized epitops

• – MabG250 reagiert nicht mit dem G250-Antigen in Western Blot-Analysen
• – die Reinigung des G250-Antigens durch MabG250-Affinitätschromatografie ist höchst ineffizient, wobei die Effizienz mit der Steigerung der für die Durchführung der Reinigung verwendeten Zeit abnimmt. Dies weist auf einen schnellen Abbau bzw. einer schnellen Entfaltung des G250-Epitops, trotz optimaler Bedingungen, zur Vermeidung von Proteolyse hin.
• – Das Hinzufügen von Detergenzien führt im Allgemeinen zu einer Senkung der G250-Nachweisgrenze in ELISA-Tests
• – Die Truncation der cDNA, die für ein G250-Antigen kodiert, gefolgt von einer transienten Transfektion in G250-negativen Zellen, gefolgt von immunhistochemischer Analyse ergab folgendes:
  
  

From general experimental evidence, it is believed that the G250 protein epitope, which is recognized by the monoclonal antibody G250 (MabG250), is a conformational epitope, most likely a non-linear epitope. This assumption is based on the following observations:

• - MabG250 does not react with the G250 antigen in Western blot analysis
• The purification of the G250 antigen by MabG250 affinity chromatography is highly inefficient, with the efficiency decreasing with the increase in time used to perform the purification. This indicates a rapid degradation or rapid unfolding of the G250 epitope, despite optimal conditions, to avoid proteolysis.
• - The addition of detergents generally leads to a lowering of the G250 detection limit in ELISA tests
• Truncation of the cDNA encoding a G250 antigen followed by transient transfection into G250 negative cells followed by immunohistochemical analysis revealed the following:
  
  

It has been demonstrated to have G250 reactivity, even when using larger cDNA constructs, get lost.

Around To further describe the G250 epitope became the NovaTope system (Novagen Inc.) uses a system that monitors the expression of all constructs, which are alloyed in this vector assures, i. all possible Fragments are expressed, regardless of the reading frame. This allows Identifying the epitopes in the middle section of a protein.

The for the MN (G250) antigen-encoding cDNA was used for 2 hours DNAse I digested to give fragments of about 50-200 nucleotides, the for Polypeptides having a length from about 15-70 amino acids have, encode. After dA tailing, the fragments were in the pScreen T-vector inserted, and the ligation mixture became the most competent E. coli DE3 bacteria added for transformation. The Bacteria were plated on selection plates. The bacterial clones were blotted on nitrocellulose filters and screened with G250 antibodies. A total of 7 clones were identified with MabG250. G250 reactivity was weak. After another screening, only one bacterial clone appeared react with MabG250, although very weak, and most likely due to background staining. Sequence analysis of the clone revealed no G250 sequences however, the presence of a truncated vector. Therefore, it turned out the dyeing of the nitrocellulose blots most likely only from a non-specific background.

In another experiment was that coding for the MN (G250) antigen digested cDNA using DNAse 1, now fragments of 200-600 Nucleotides for Encode polypeptides of about 65-200 amino acids, were isolated and cloned into the expression vector and were expressed in bacteria. Different bacterial colonies were on G250 reactivity screened.

A Reactivity with G250 was not apparent. The blots were over a long time stained, after which a weak staining of 6 bacterial isolates was observed. The bacterial isolates contained only a truncated vector and an unrelated sequence.

As a result, failed to sequence the MN antigen with strong reactivity to MabG250 being found.

These Results strongly suggest that the G250 epitope is a conformational Epitope is. Furthermore the G250 epitope on the MN antigen can not be detected by conventional Procedures, even after considerable effort, are characterized.

Example 3

Development of a chimera G250 IgG producing cell line

General strategy

Around a murine / human chimera version of the mouse G250 antibody To construct, the genes of the heavy variable region became and light chains that determine the binding specificity of the antibody from the G250 mouse hybridoma cloned and assembled in vitro with the genes for the human constant region, to murine / human chimeric antibody genes to build. The expression of these chimeric genes in the appropriate Cell line led for making a chimeric antibody with the same binding properties of the original mouse antibody, wherein but about 75% of the molecule include human sequences.

The Strategy for cloning the variable regions of the genes for the severe and light chain of the G250 hybridoma was based on the compound in the genome between the variable region and the corresponding one J (joining) region around a functional rearrangement (and expression) to obtain the immunoglobulin genes. DNA sands for the J region can be used for Screening genome libraries used to create a DNA to isolate which is coupled to the J regions.

The DNA in the germline configuration (i.e., not rearranged) hybridizes also on J samples, but is not due to a sequence of the variable region coupled and can thus be isolated by restriction enzyme analysis Clones are identified.

The cloning strategy was therefore used to isolate variable regions of non-rearranged heavy and light chain genes by JH and JK probes. Further, cDNA clones corresponding to the heavy and light chain variable regions were also derived from the G250 Hyb obtained mRNA to support the identification and characterization of the correct genome clones. These genomic clones, which match the G250 cDNA sequences, were cloned into expression vectors containing the human constant regions and transfected into mouse myeloma cells to determine if an antibody was produced. The antibody of the producing cells was then tested for binding specificity compared to the mouse G250 antibody.

Cloning of the G250 VH and VL cDNA

Total RNA was isolated from the G250 hybridoma cell and used to make specific VH and VL cDNA clones. The "first-strand cDNA synthesis" was performed using oligonucleotide primers specific for murine kappa and IgG-constant regions After dG tailing, "second-strand synthesis" was performed using C-terminus. Tailored oligonucleotide primers performed. The heavy and light chain variable regions were amplified by polymerase chain reaction using specific 5 'and 3' primers and the amplification products were cloned into the plasmid vector pUC19. VN and VL clones were subjected to DNA sequence analysis to determine the base sequences of the putative heavy and light chain G250 variable regions. The results of the sequencing are in 1 shown.

Cloning of the genes of variable G250 region

Around To clone the genes of the variable region was genomic High molecular weight DNA from the G250 hybridoma cells by means of Treatment of isolated cell nuclei with SDS and proteinase K, and followed by isolated from two extractions with phenol and a precipitate with ethanol.

Southern blot analysis using a probe for the J region for the heavy chain gene locus indicates that the G250 heavy chain was contained on a 2.3 kilobase (Kb) Eco RI DNA fragment. Accordingly, Eco RI fragments of the hybridoma DNA were fractionated on a 0.8% agarose gel and fragments of the size range of about 2-3 Kb were eluted from the gel and ligated to the vector arms of a lambda bacteriophage vector Zap II (Stratagene , La Jolla California, USA). The ligation was trapped in phage particles using Gigapack Gold (Stratagene) in vitro and plated on E. coli LE392 cells at a density of approximately 20,000 colonies per 150 mm plate. The colonies were transferred to nitrocellulose filters and probed with a ³² P-labeled DNA probe (a 2.0 Kb Eco RI-Bam HI fragment containing the murine J3 and J4 exons).

For the G250 light chain variable region genes, Southern blot analysis using a murine probe for the J region of the kappa locus revealed that the correct gene was positioned on a 5.5 Kb HindIII fragment. Accordingly, the G250 hybridoma DNA was digested with HindIII and DNA fragments of 5-6 Kb were isolated from a 0.8% agarose gel and ligated to the arms of the bacteriophage lambda vector Charon 27. The ligated DNA was digested in phage particles using Gigapack Gold (Stratagene) in vitro and plated on E. coli LE392 cells at a density of approximately 20,000 colonies per 150 mm plate. The colonies were transferred to nitrocellulose filters, and a ³² P-labeled DNA Sondesondiert (a 2.7Kb HindIII fragment containing all five J kappa exons).

positive Clones for the genes of the heavy and light chain were after at least 3 cleaning passes of the colonies isolated using the J region probe to check the hybridization to the phage DNA at each purification step. purified DNA from the isolated phage clones for the genes of the Light chain was labeled with Eco RI (heavy chain) or HindIII (light chain) Chain) digested and fractionated on agarose gels. The suitable Size of the fragments (2.3 Kb for the heavy chain and 5.5 Kb for the light chain) were isolated from the gels and into the plasmid vector subcloned pUC19. By restriction endonuclease mapping and DNA sequence analysis has been shown to produce these subcloned DNA fragments the genes of the variable regions of the antibody corresponding to the structures of the cDNA clones originally from the G250 hybridoma cell.

expression

The 2.3 Kb Eco RI heavy chain variable region fragment was cloned into a suitable expression vector containing the human constant G1 region and a gene conferring resistance to a selection marker in mammalian cells. The 5.5 Kb HindIII light chain fragment was cloned into a suitable expression vector containing the human kappa constant region and a gene conferring resistance to a selection marker in mammalian cells. The presence of cloning fragments and their orientation in the expression vectors was determined by restriction enzyme mapping. The basic vectors were previously used to make chimeric antibodies (Sun, L. et al., (1987) PNAS 84, page 214, Knight et al., (1993), Molecular Immunology 30, pages 1443-1453). Expression of the genes of the chimeric antibody is achieved using the natural immunoglobulin promoters upstream of the cloned variable regions. Downstream regulatory signals are provided by elements related to the constant region genes.

Expression of chimeric G250 antibody

The Heavy and light chain expression vectors were used using the electroporation technique a non-producing Mouse myeloma cell line to co-transfect. An ELISA assay was used to clones on the antibody, which includes the humane Fc region, and the most productive one Clone (cG250) was used for the further characterization is selected.

The chimeric antibody was purified by chromatography on protein A-Sepharose and analyzed in a competition assay to verify that the antibody has the correct specificity. It has been demonstrated that the cG250 antibody is as effective in competing for binding to A704 renal carcinoma cells (expressing the G250 antigen) with the ¹²⁵ I-labeled mouse G250 antibody as the unlabelled mouse G250 antibody. Therefore, the chimeric G250 antibody has similar binding properties as the original mouse G250 antibody.

Example 4

Use of chimeric G250 antibody in a clinical trial

administration protocol

patients with renal cell carcinoma having metastases after surgery, were administered by administering a 50 minute chimeric G250 antibody infusion for 30 minutes once per week for 12 weeks, treated.

Results of effectiveness:

In The present study was performed in eight out of 32 patients (25% of patients) Patients) reached a persistent stable illness for six months. The average Time of progression of the disease was 16 weeks for all patients (Mean 26.67 weeks) within a range of 4 to 70 Weeks. Since the deadline, December 2001, is five patients still no disease progression occurred (51+, 56+, 60+, 64+ and 70+ weeks).

The Importance of these findings is supported by the fact that Most of these patients had a proven at baseline Progression of the disease and for another two patients Progression was suspected. In addition, three of these patients had Renitence opposite an earlier one Cytokine and chemo on or had following a Cytokine treatment after several years a relapse. Four patients were previously undergone no treatment and were tested with the test agent treated immediately after diagnosis of metastases. At the beginning of the study Three of these patients had a Karnofsky performance status (KPS) of 80%, two a 90% KPS. The hemoglobin value (Hgb) was at 5 patients under 10 g / dL. The mean performance status of 90% (mean 91%) is equal to that of the patient groups receiving the treatment not struck. The mean Hgb value of 9.8 g / dl (mean 10.97 g / dl) is also relatively low.

One Patient achieved a complete objective remission. The objective response came late, more than six months after the start of the therapy study. Another patient showed a significant decline 59% of its size Target lesions. The response of both patients lasts week 64 and week respectively 70, as assessed in December 2001. The tumor decline occurred late, more than six months after the start of the therapy study. An explanation of this late Response may be due to the duration of treatment with intravenous injections of the chimera antibody was relatively short and a late one Immune response not with ADCC development related.

The response lasts more than a year and continues in December 2001. The overall size the clinical benefits (at least more than six months or an objective response) is 25%. In addition, it should be noted that six months after the start of the treatment trial, 87.5% (28/32) of the patients were still alive.

The End November 2001 was an estimate the median survival calculated at least 13.5 months.

The End January 2002 was an estimate the median survival calculated at least 15 months. This is significantly higher than the median chance of survival of untreated patients (3 months) and as the median chance of survival patients treated with FDA-approved therapy were, i. by administering a high dose of interleukin 2 (10-12 Months).

It Thus, it has been demonstrated that the chimeric antibody G250 is a suitable therapeutic agent Funds for the treatment of renal cell carcinoma is. In a preferred embodiment Treatment includes surgical removal of the primary tumor and a subsequent one Administration of G250 to the metastases that spread throughout the body have to turn off.

Claims (14)

Hybridoma cell DSM ACC 2526, the monoclonal antibody G250 produced. Use of the hybridoma cell according to claim 1 for the preparation a progeny cell carrying the monoclonal antibody G250 produced. Use according to claim 2, wherein the progeny cell by transfer of genetic material, which for the antibody Encodes G250 or at least one antigen-binding site thereof, has been obtained in a receptor cell. Use according to claim 2 or 3, wherein the progeny cell produces a chimeric antibody, a humanized antibody, a fully humanized antibody, a bispecific antibody, a single-chain antibody or F (ab ') ₂ , Fab' or Fab antibody fragments. Use according to one the claims 2 to 4, wherein the progeny cell is a prokaryotic cell. Use according to claim 5, wherein the progeny cell is an E. coli cell. Use according to one the claims 2 to 4, wherein the progeny cell is a eukaryotic cell. Use according to claim 7, wherein the progeny cell is a yeast cell, a myeloma cell or a CHO cell is. Method for producing an antibody G250, comprising: culturing a cell according to any one of claims 1-7 suitable conditions in which the antibody is produced and the Obtaining the antibody from the cell and / or from the culture medium. Method according to claim 9 for use for the preparation of a pharmaceutical composition containing the antibody G250 as an active ingredient. Method according to claim 10, wherein the pharmaceutical composition comprises the chimeric antibody G250 includes. Method according to claim 10, wherein the pharmaceutical composition comprises the chimeric antibody G250 in radiolabelled form. Method according to claim 10, wherein the pharmaceutical composition comprises a chimeric antibody G250, to a cytokine, such as IL-2, TNF and / or GM-CSF, is bound. Use of a cell according to any one of claims 1-7 for the preparation an antibody G250.